U.S. patent number 9,272,069 [Application Number 12/864,045] was granted by the patent office on 2016-03-01 for adhesive complex coacervates and methods of making and using thereof.
This patent grant is currently assigned to University of Utah Research Foundation. The grantee listed for this patent is Hui Shao, Russell Stewart. Invention is credited to Hui Shao, Russell Stewart.
United States Patent |
9,272,069 |
Stewart , et al. |
March 1, 2016 |
Adhesive complex coacervates and methods of making and using
thereof
Abstract
Described herein is the synthesis of adhesive complex
coacervates. The adhesive complex coacervates are composed of a
mixture of one or more polycations, one or more polyanions, and one
of more multivalent cations. The polycations and polyanions in the
adhesice complex coacervate are crosslinked with one another by
covalent bonds upon curing. The adhesive complex coacervates have
several desirable features when compared to conventional
bioadhesives, which are effective in water-based applicatgions. The
adhesive complex coacervates described herein exhibit good
interfacial tension in water when applied to a substrate (i.e.,
they spread over the interface rather than being beaded up).
Additionally, the ability of the complex coacervate to crosslink
intermolecularly increases the cohesive strength of the adhesive
complex coacervate. The adhesive complex coacervates have numerous
biological applications as bioadhesives and drug delivery devices.
In particular, the adhesive complex coacervates described herein
are particularly useful in underwater applications and situations
where water is present such as, for example, physiological
conditions.
Inventors: |
Stewart; Russell (Salt Lake
City, UT), Shao; Hui (Salt Lake City, UT) |
Applicant: |
Name |
City |
State |
Country |
Type |
Stewart; Russell
Shao; Hui |
Salt Lake City
Salt Lake City |
UT
UT |
US
US |
|
|
Assignee: |
University of Utah Research
Foundation (Salt Lake City, UT)
|
Family
ID: |
40901376 |
Appl.
No.: |
12/864,045 |
Filed: |
November 13, 2008 |
PCT
Filed: |
November 13, 2008 |
PCT No.: |
PCT/US2008/083311 |
371(c)(1),(2),(4) Date: |
July 22, 2010 |
PCT
Pub. No.: |
WO2009/094060 |
PCT
Pub. Date: |
July 30, 2009 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20100305626 A1 |
Dec 2, 2010 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61023173 |
Jan 24, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C08F
8/00 (20130101); C09J 133/14 (20130101); A61L
24/0015 (20130101); A61L 27/54 (20130101); A61L
24/043 (20130101); C08F 8/10 (20130101); A61L
27/26 (20130101); C08F 8/06 (20130101); C08F
8/10 (20130101); C08F 230/02 (20130101); C08F
8/00 (20130101); C08F 230/02 (20130101); C08F
8/06 (20130101); C08F 230/02 (20130101); C08F
2810/20 (20130101); C08F 2800/10 (20130101); C08F
2810/30 (20130101); A61L 2300/412 (20130101); A61L
2300/602 (20130101); A61L 2300/62 (20130101) |
Current International
Class: |
C08F
230/02 (20060101); A61L 27/54 (20060101); C08F
8/06 (20060101); C08F 8/00 (20060101); C09J
133/14 (20060101); A61L 24/00 (20060101); A61L
24/04 (20060101); A61L 27/26 (20060101) |
References Cited
[Referenced By]
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CN |
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EP |
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WO |
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May 2012 |
|
WO |
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|
Primary Examiner: Salamon; Peter A
Attorney, Agent or Firm: Gardner Groff Greenwald &
Villanueva, PC
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority upon U.S. provisional application
Ser. No. 61/023,173, filed Jan. 24, 2008. This application is
hereby incorporated by reference in its entirety for all of its
teachings.
Claims
What is claimed:
1. An adhesive comprising a complex coacervate, wherein the complex
coacervate comprises at least one synthetic polycation, at least
one synthetic polyanion, and at least one multivalent cation,
wherein the synthetic polycation and the synthetic polyanion each
comprise at least one crosslinkable group covalently bonded to the
synthetic polycation and the synthetic polyanion capable of
covalently crosslinking with one another.
2. The coacervate of claim 1, wherein the polycation comprises a
polyamino compound.
3. The coacervate of claim 1, wherein the polycation comprises a
polyacrylate comprising one or more pendant amino groups.
4. The coacervate of claim 1, wherein the polycation comprises a
polyacrylate comprising one or more pendant imidazole groups.
5. The coacervate of claim 1, wherein the polycation comprises a
polymer comprising at least one fragment comprising the formula I
##STR00003## wherein R.sup.1, R.sup.2, and R.sup.3 are,
independently, hydrogen or an alkyl group, X is oxygen or NR.sup.5,
where R.sup.5 is hydrogen or an alkyl group, and m is from 1 to 10,
or the pharmaceutically-acceptable salt thereof.
6. The coacervate of claim 5, wherein R.sup.1, R.sup.2, and R.sup.3
are methyl, X is NH, and m is 2.
7. The coacervate of claim 2, wherein the polyamino compound
comprises from 10 to 90 mole % primary, secondary, or tertiary
amino groups.
8. The coacervate of claim 1, wherein the polyanion comprises one
or more sulfate, sulfonate, carboxylate, borate, boronate,
phosphonate, or phosphate groups.
9. The coacervate of claim 1, wherein the polyanion comprises a
polyphosphate compound.
10. The coacervate of claim 1, wherein the polyanion comprises a
polyacrylate comprising one or more pendant phosphate groups.
11. The coacervate of claim 1, wherein the polyanion comprises a
polymer comprising at least one fragment comprising the formula II
##STR00004## wherein R.sup.4 is hydrogen or an alkyl group, and n
is from 1 to 10, or the pharmaceutically-acceptable salt
thereof.
12. The coacervate of claim 11, wherein R.sup.4 is methyl and n is
2.
13. The coacervate of claim 10, wherein the polyphosphate compound
comprises from 10 to 90 mole % phosphate groups.
14. The coacervate of claim 1, wherein the polyanion comprises a
polyphosphoserine.
15. The coacervate of claim 1, wherein the multivalent cation
comprises one or more transition metal ions or rare earth
metals.
16. The coacervate of claim 1, wherein the multivalent cation
comprises one or more divalent cations.
17. The coacervate of claim 1, wherein the multivalent cation
comprises Ca.sup.+2 and Mg.sup.+2.
18. The coacervate of claim 1, wherein the composition is
biocompatible and biodegradable.
19. The coacervate of claim 1, wherein the composition further
comprises one or more bioactive agents encapsulated in the
complex.
20. The coacervate of claim 1, wherein the groups capable of
crosslinking with one another are the same or different.
21. The coacervate of claim 1, wherein the crosslinking group on
the polycation comprises a nucleophilic group and the crosslinking
group on the polyanion comprises an electrophilic group.
22. The coacervate of claim 1, wherein the crosslinking group on
the polycation and polyanion comprises a hydroxyl aromatic compound
capable of undergoing oxidation.
23. The coacervate of claim 1, wherein the crosslinking group on
the polyanion comprises a DOPA residue or a catechol residue and
the polycation comprises a nucleophilic group capable of reacting
with the crosslinking group to form a covalent bond.
24. An adhesive complex coacervate produced by the process
comprising (a) preparing a polyelectrolyte complex comprising
admixing at least one synthetic polycation, at least one synthetic
polyanion, and at least one multivalent cation, and the synthetic
polycation and synthetic polyanion each comprise at least one group
capable of covalently crosslinking with each other; and (b)
adjusting the pH of the polyelectrolyte complex, the concentration
of the at least one divalent cation, or a combination thereof to
produce the adhesive complex coacervate.
25. The coacervate of claim 24, wherein the coacervate is produced
in situ.
26. The coacervate of claim 24, wherein step (a) is performed at a
pH less than 4, and step (b) comprises raising the pH.
27. The coacervate of claim 24, wherein step (b) comprises raising
the pH of the polyelectrolyte complex to a pH of greater than or
equal to 7.0.
28. The coacervate of claim 24, wherein after step (b) further
comprises contacting the complex coacervate with an oxidant in
order to facilitate the crosslinking between the polycation and
polyanion.
29. The coacervate of claim 28, wherein the oxidant comprises
O.sub.2, NaIO.sub.4, a peroxide, or a transition metal oxidant.
30. A method for repairing a bone fracture in a subject, comprising
contacting the fractured bone with the adhesive complex coacervate
of claim 1 and covalently crosslinking the synthetic polycation and
synthetic polyanion in the adhesive complex coacervate.
31. The method of claim 30, wherein the method is in vitro.
32. The method of claim 30, wherein the method is in vivo.
33. The method of claim 30, wherein the fracture comprises complete
fracture, an incomplete fracture, a linear fracture, a transverse
fracture, an oblique fracture, a compression fracture, a spiral
fracture, a comminuted fracture, a compacted fracture, or an open
fracture.
34. The method of claim 30, wherein the fracture comprises an
intra-articular fracture.
35. The method of claim 30, wherein the fracture comprises a
craniofacial bone fracture.
36. The method of claim 30, wherein the method comprises adhering a
fractured piece of bone to an existing bone.
37. The method of claim 30, wherein the composition sets within 60
seconds.
38. A method for adhering a metal substrate to a bone of a subject
comprising contacting the bone with the composition of claim 1,
applying the metal substrate to the coated bone, and covalently
crosslinking the synthetic polycation and synthetic polyanion in
the adhesive complex coacervate.
39. A method for adhering a bone-tissue scaffold to a bone of a
subject comprising contacting the bone and tissue with the
composition of claim 1, applying the bone-tissue scaffold to the
bone and tissue, and covalently crosslinking the synthetic
polycation and synthetic polyanion in the adhesive complex
coacervate.
40. The method of claim 39, wherein the tissue comprises cartilage,
a ligament, a tendon, a soft tissue, an organ, or synthetic
derivative thereof.
41. The method of claim 39, wherein the scaffold comprises one or
more drugs that facilitate growth or repair of the bone and
tissue.
42. A method for repairing a crack in a tooth, comprising applying
the composition of claim 1 to the crack and covalently crosslinking
the synthetic polycation and synthetic polyanion in the adhesive
complex coacervate.
43. A method for securing a dental implant, comprising applying the
composition of claim 1 to an oral substrate, attaching the dental
implant to the substrate, and covalently crosslinking the synthetic
polycation and synthetic polyanion in the adhesive complex
coacervate.
44. The method of claim 43, wherein the dental implant comprises a
crown or denture.
45. A method for delivering one or more bioactive agents comprising
administering the coacervate of claim 1 to a subject.
46. The coacervate of claim 1, wherein the polycation comprises a
polyamino compound and the polyanion comprises a polyphosphate
compound.
Description
BACKGROUND
Bone fractures are a serious health concern in society today. In
addition to the fracture itself, a number of additional health
risks are associated with the fracture. For example,
intra-articular fractures are bony injuries that extend into a
joint surface and fragment the cartilage surface. Fractures of the
cartilage surface often lead to debilitating posttraumatic
arthritis. The main determining factors in the development of
posttraumatic arthritis are thought to be the amount of energy
imparted at the time of injury, the patient's genetic
predisposition (or lack thereof) to posttraumatic arthritis, and
the accuracy and maintenance of reduction. Of the three prognostic
factors, the only factor controllable by orthopedic caregivers is
achievement and maintenance of reduction. Comminuted injuries of
the articular surface (the cartilage) and the metaphysis (the
portion of the bone immediately below the cartilage) are
particularly challenging to maintain in reduced (aligned) position.
This relates to the quality and type of bone in this area. It also
relates to the limitations of fixation with titanium or stainless
steel implants.
Currently, stainless steel and titanium implants are the primary
methods of fixation, but their size and the drilling necessary to
place them frequently interfere with the exact manipulation and
reduction of smaller pieces of bone and cartilage. A variety of
bone adhesives have been tested as alternatives to mechanical
fixation. These fall into four categories: polymethylmethacrylates
(PMMA), fibrin-based glues, calcium phosphate (CP) cements, and CP
resin composites. PMMA cements, which are used in the fixation of
protheses, have well-known drawbacks, one of the most serious being
that the heat generated from the exothermic setting reaction can
kill adjacent bone tissue. Also, the poor bonding to bone leads to
aseptic loosening, the major cause of PMMA cemented prothesis
failure.
Fibrin glues, based on the blood clotting protein fibrinogen, have
been tested for fixing bone grafts and repairing cartilage since
the 1970s and yet have not been widely deployed. One of the
drawbacks of fibrin glues is that they are manufactured from pooled
human donor blood. As such, they carry risk of transmitting
infections and could potentially be of limited supply.
CP cements are powders of one or more forms of CP, e.g.,
tetracalcium phosphate, dicalcium phosphate anhydride, and
.beta.-tricalcium phosphate. When the powder is mixed with water it
forms a paste that sets up and hardens through the entanglement of
one or more forms of CP crystals, including hydroxyapatite.
Advantages of CP cements include isothermal set, proven
biocompatibility, osteoconductivity, and they serve as a reservoir
for Ca and PO.sub.4 for hydroxyapatite formation during healing.
The primary disadvantages are that CP cements are brittle, have low
mechanical strength and are therefore not ideal for stable
reduction of small articular segments. CP cements are used mostly
as bone void fillers. The poor mechanical properties of CP cements
have led to composite cements of CP particles and polymers. By
varying the volume fractions of the particulate phase and the
polymer phase, the modulus and strength of the glue can be adjusted
toward those of natural bone, an avenue that is also open to
us.
Given the overall health impact associated with bone fractures and
the imperfect state of current fixation methods, new fixation
methods are needed.
SUMMARY
Described herein is the synthesis of adhesive complex coacervates.
The adhesive complex coacervates are composed of a mixture of one
or more polycations, one or more polyanions, and one of more
multivalent cations. The polycations and polyanions are crosslinked
with one another by covalent bonds upon curing. The adhesive
complex coacervates have several desirable features when compared
to conventional adhesives, which are effective in water-based
applications. The adhesive complex coacervates described herein
exhibit low interfacial tension in water when applied to a
substrate (i.e., they spread over the interface rather than being
beaded up). Additionally, the ability of the complex coacervate to
crosslink intermolecularly increases the cohesive strength of the
adhesive complex coacervate. The adhesive complex coacervates have
numerous biological applications as bioadhesives and drug delivery
devices. In particular, the adhesive complex coacervates described
herein are particularly useful in underwater applications and
situations where water is present such as, for example,
physiological conditions.
The advantages of the invention will be set forth in part in the
description which follows, and in part will be obvious from the
description, or may be learned by practice of the aspects described
below. The advantages described below will be realized and attained
by means of the elements and combinations particularly pointed out
in the appended claims. It is to be understood that both the
foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated in and constitute
a part of this specification, illustrate several aspects described
below.
FIG. 1 shows a model of pH dependent coacervate structure and
adhesive mechanisms. (A) The polyphosphate (black) with low charge
density paired with the polyamine (red) form nm-scale complexes.
The complexes have a net positive charge. (B) Extended high charge
density polyphosphates form a network connected by more compact
lower charge density polyamines and when present divalent cations
(green symbols). The net charge on the copolymers is negative. (C)
Oxidation of 3,4-dihydroxyphenol (D) by O.sub.2 or an added oxidant
initiates crosslinking between the quinone (Q) and primary amine
sidechains. The coacervate can adhere to the hydroxyapatite surface
through electrostatic interactions, 3,4-dihydroxyphenol sidechains,
and quinone-mediated covalent coupling to matrix proteins.
FIGS. 2-7 shows several protein sequences produced by P.
californica that can be used as polycations and polyanions in the
present invention as well as synthetic polycations and polyanions
useful in the present invention.
FIG. 8 shows different mechanisms of DOPA crosslinking.
FIG. 9 shows dual syringe systems for applying small "spot welds"
of complex coacervates described herein to repair fractures (A),
small bone injuries (B), or bonding synthetic scaffolds to bony
tissue (C).
FIG. 10 shows the structure and UV/VIS characterization of mimetic
copolymers. (A) The Pc3 analog, 1, contained 88.4 mol % phosphate,
9.7 mol % dopamide, and 0.1 mol % FITC sidechains. The Pc1 analog,
2, contained 8.1 mol % amine sidechains. The balance was acrylamide
subunits in both cases. (B) A single peak at 280 nm characteristic
of the catechol form of 3,4-dihydroxyphenol was present in the
spectrum of 1. Following oxidation with NaIO.sub.4 a peak at 395 nm
corresponding to the quinone form appeared confirming the expected
redox behavior of the 3,4-dihydroxyphenol containing polymer.
FIG. 11 shows the pH dependent complex coacervation of mixed
polyelectrolytes. (A) At low pH, a 50 mg/ml mixture of 1 and 2
having equal quantities of amine and phosphate sidechains formed
stable colloidal PECs. As the pH increased the polymers condensed
into a dense liquid complex coacervate phase. At pH 10 the
copolymers went into solution and oxidatively crosslinked into a
clear hydrogel. (B) The net charge of the copolymer sidechains as a
function of pH calculated from the copolymer sidechain densities.
(C) The diameter of the PECs (circles) increased nearly three-fold
over the pH range 2-4. Above pH 4 the complexes flocculate and
their size could not be measured. The zeta potential (squares) was
zero near pH 3.6 in agreement with the calculated net charge.
FIG. 12 shows the liquid character of an adhesive complex
coacervate. The solution of 1 and 2 contained equal quantities of
amine and phosphate sidechains, pH 7.4.
FIG. 13 shows the phase diagram of polyelectrolytes and divalent
cations. The amine to phosphate sidechain and phosphate sidechain
to divalent cation ratios were varied at a fixed pH 8.2. The state
of the solutions represented in a gray scale. The mass (mg) of the
coacervate phase is indicated in the dark grey squares. The
compositions indicated with an asterisk were used to test bond
strength.
FIG. 14 shows the bond strength, shear modulus, and dimensional
stability of coacervate bonded bones. (A) Bond strength at failure
increased .about.50% and the stiffness doubled as the divalent
cation ratio went from 0 to 0.4 relative to phosphate sidechains.
Specimens wet bonded with a commercial cyanoacrylate adhesive were
used as a reference. (n=6 for all conditions) (B) Bonds of adhered
bone specimens fully submerged in PBS for four months (pH 7.2) did
not swell appreciably.
FIG. 15 shows UV-vis spectra of dopamine copolymers before and
after oxidation (pH 7.2). A catechol peak present before oxidation
was converted into the quinone form. Top left: p(DMA[8]-Aam[92]).
Bottom left: p(AEMA[30]-DMA[8]). Right: Hydrogel formation by
oxidative crosslinking of dopamine copolymers. (A)
p(DMA[8]-Aam[92]). (B) p(EGMP[92]-DMA[8]). (C) p(DMA[8]-Aam[92])
mixed with p(AEMA[30]-Aam[70]). (D) p(EGMP[92]-DMA[8]) mixed with
p(AEMA[30]-Aam[70]). Bracketed numbers indicate mol % of
sidechains. Arrows indicate direction spectra are changing over
time.
FIG. 16 shows pH dependence of dopamine oxidation in
poly(EGMP[92]-DMA[8]). Arrows indicate direction spectra change
with time. Top: pH 5.0, time course inset. Bottom: pH 6.0.
FIG. 17 shows direct contact of (A) human foreskin fibroblasts, (B)
human tracheal fibroblasts, and (C) rat primary astrocytes with
adhesive (red auto-fluorescent chunks, white asterisks). Cell
morphology, fibronectin secretion, and motility are
indistinguishable from cells growing in the absence of glue.
Green=intermediate filament proteins. Red=secreted fibronection.
Blue=DAPI stained nuclei.
FIG. 18 shows a multi-fragment rat calvarial defect model. (A)
Generation of defect. (B) Fragmentation of bone cap. (C)
Replacement of fragments in defect. (D) Application of bone glue.
(E-F) Curing (darkening) of glue. Fragments are firmly fixed in E
and F.
FIG. 19 shows the effect of pH and normalized net charge with
respect to forming adhesive complex coacervates.
DETAILED DESCRIPTION
Before the present compounds, compositions, articles, devices,
and/or methods are disclosed and described, it is to be understood
that the aspects described below are not limited to specific
compounds, synthetic methods, or uses as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular aspects only and is not
intended to be limiting.
In this specification and in the claims that follow, reference will
be made to a number of terms that shall be defined to have the
following meanings:
It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of two or more such carriers, and the like.
"Optional" or "optionally" means that the subsequently described
event or circumstance can or cannot occur, and that the description
includes instances where the event or circumstance occurs and
instances where it does not. For example, the phrase "optionally
substituted lower alkyl" means that the lower alkyl group can or
can not be substituted and that the description includes both
unsubstituted lower alkyl and lower alkyl where there is
substitution.
Ranges may be expressed herein as from "about" one particular
value, and/or to "about" another particular value. When such a
range is expressed, another aspect includes from the one particular
value and/or to the other particular value. Similarly, when values
are expressed as approximations, by use of the antecedent "about,"
it will be understood that the particular value forms another
aspect. It will be further understood that the endpoints of each of
the ranges are significant both in relation to the other endpoint,
and independently of the other endpoint.
References in the specification and concluding claims to parts by
weight, of a particular element or component in a composition or
article, denotes the weight relationship between the element or
component and any other elements or components in the composition
or article for which a part by weight is expressed. Thus, in a
compound containing 2 parts by weight of component X and 5 parts by
weight component Y, X and Y are present at a weight ratio of 2:5,
and are present in such ratio regardless of whether additional
components are contained in the compound.
A weight percent of a component, unless specifically stated to the
contrary, is based on the total weight of the formulation or
composition in which the component is included.
Variables such as R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, X,
m, and n used throughout the application are the same variables as
previously defined unless stated to the contrary.
The term "alkyl group" as used herein is a branched or unbranched
saturated hydrocarbon group of 1 to 25 carbon atoms, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl,
pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl,
eicosyl, tetracosyl and the like. Examples of longer chain alkyl
groups include, but are not limited to, an oleate group or a
palmitate group. A "lower alkyl" group is an alkyl group containing
from one to six carbon atoms.
Any of the compounds described herein can be the
pharmaceutically-acceptable salt. In one aspect,
pharmaceutically-acceptable salts are prepared by treating the free
acid with an appropriate amount of a pharmaceutically-acceptable
base. Representative pharmaceutically-acceptable bases are ammonium
hydroxide, sodium hydroxide, potassium hydroxide, lithium
hydroxide, calcium hydroxide, magnesium hydroxide, ferrous
hydroxide, zinc hydroxide, copper hydroxide, aluminum hydroxide,
ferric hydroxide, isopropylamine, trimethylamine, diethylamine,
triethylamine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, 2-diethylaminoethanol, lysine, arginine,
histidine, and the like. In one aspect, the reaction is conducted
in water, alone or in combination with an inert, water-miscible
organic solvent, at a temperature of from about 0.degree. C. to
about 100.degree. C. such as at room temperature. In certain
aspects where applicable, the molar ratio of the compounds
described herein to base used are chosen to provide the ratio
desired for any particular salts. For preparing, for example, the
ammonium salts of the free acid starting material, the starting
material can be treated with approximately one equivalent of
pharmaceutically-acceptable base to yield a neutral salt.
In another aspect, if the compound possesses a basic group, it can
be protonated with an acid such as, for example, HCl, HBr, or
H.sub.2SO.sub.4, to produce the cationic salt. In one aspect, the
reaction of the compound with the acid or base is conducted in
water, alone or in combination with an inert, water-miscible
organic solvent, at a temperature of from about 0.degree. C. to
about 100.degree. C. such as at room temperature. In certain
aspects where applicable, the molar ratio of the compounds
described herein to base used are chosen to provide the ratio
desired for any particular salts. For preparing, for example, the
ammonium salts of the free acid starting material, the starting
material can be treated with approximately one equivalent of
pharmaceutically-acceptable base to yield a neutral salt.
Described herein are adhesive complex coacervates and their
applications thereof. In general, the complexes are a mixture of
cations and anions in balanced proportions to produce stable
aqueous complexes at a desired pH. The adhesive complex coacervate
comprises at least one polycation, at least one polyanion, and at
least one multivalent cation, wherein at least one polycation or
polyanion is a synthetic compound, and the polycation and/or
polyanion are crosslinked with one another upon curing the complex
coacervate. Each component of the coacervate and methods for making
the same are described below.
The adhesive complex coacervate is an associative liquid with a
dynamic structure in which the individual polymer components
diffuse throughout the entire phase. Complex coacervates behave
rheologically like viscous particle dispersions rather than a
viscoelastic polymer solution. As described above, the adhesive
complex coacervates exhibit low interfacial tension in water when
applied to substrates either under water or that are wet. In other
words, the complex coacervate spreads evenly over the interface
rather than being beading up. Additionally, upon intermolecular
crosslinking, the adhesive complex coacervate forms a strong,
insoluble, cohesive material.
Conversely, polyelectrolyte complexes (PECs), which can be a
precursor to the adhesive complex coacervates described herein, are
small colloidal particles. For example, referring to FIG. 11A, a
solution of PECs at pH 3.1 and 4.2 exists as a milky solution of
colloidal particles having a diameter of about 300 nm Upon raising
the pH to 7.2 and 8.1, the PEC condenses into a liquid phase of
concentrated polymers (the coacervate phase) and a dilute
equilibrium phase. In this aspect, the PEC can be converted to an
adhesive complex coacervate described herein.
An exemplary model of the differences in phase behavior between the
polyelectrolyte complex and the adhesive complex coacervate is
presented in FIG. 1. At low pH the oppositely charged
polyelectrolytes associate electrostatically into nano-complexes
with a net positive surface charge that stabilizes the suspension
to produce PEC 1. With increasing pH the net charge of the
complexes changes from positive to negative but remains near net
neutrality. The PEC can form a loose precipitate phase, which can
be converted to a complex coacervate 2 by raising the pH further
(FIG. 1). Thus, in certain aspects, the conversion of the PEC to
complex coacervate can be "triggered" by adjusting the pH and/or
the concentration of the multivalent cation. For example, the PEC
can be produced at a pH of less than or equal to 4, and the pH of
the PEC can be raised to greater than or equal to 7.0, from 7.0 to
9.0, or from 8.0 to 9.0 to convert the PEC to a complex coacervate.
Subsequent crosslinking between the polycation and polyanions
(e.g., oxidation and covalent crosslinking as shown in FIG. 1C)
results in the formation of the adhesive complex coacervate
described herein.
The polycations and polyanions contain groups that permit
crosslinking between the two polymers upon curing to produce new
covalent bonds and the adhesive complex coacervate described
herein. The mechanism of crosslinking can vary depending upon the
selection of the crosslinking groups. In one aspect, the
crosslinking groups can be electrophiles and nucleophiles. For
example, the polyanion can have one or more electrophilic groups,
and the polycations can have one or more nucleophilic groups
capable of reacting with the electrophilic groups to produce new
covalent bonds. Examples of electrophilic groups include, but are
not limited to, anhydride groups, esters, ketones, lactams (e.g.,
maleimides and succinimides), lactones, epoxide groups, isocyanate
groups, and aldehydes. Examples of nucleophilic groups are
presented below.
In one aspect, the crosslinkable group includes a
hydroxyl-substituted aromatic group capable of undergoing oxidation
in the presence of an oxidant. In one aspect, the
hydroxyl-substituted aromatic group is a dihydroxyphenol or
halogenated dihydroxyphenol group such as, for example, DOPA and
catechol (3,4 dihydroxyphenol). For example, in the case of DOPA,
it can be oxidized to dopaquinone. Dopaquinone is an electrophilic
group that is capable of either reating with a neighboring DOPA
group or another nucleophilic group. In the presence of an oxidant
such as oxygen or other additives including, but not limited to,
peroxides, periodates, or transition metal oxidants (e.g.,
NaIO.sub.4 or a Fe.sup.+3 compound), the hydroxyl-substituted
aromatic group can be oxidized. In another aspect, crosslinking can
occur between the polycation and polyanion via light activated
crosslinking through azido groups. Once again, new covalent bonds
are formed during this type of crosslinking.
The stability of the oxidized crosslinker can vary. For example,
the phosphono containing polyanions described herein that contain
oxidizable crosslinkers are stable in solution and do not crosslink
with themselves. This permits nucleophilic groups present on the
polycation to react with the oxidized crosslinker. This is a
desirable feature of the invention, which permits the formation of
intermolecular bonds and, ultimately, the formation of a strong
adhesive. Examples of nucleophilic groups that are useful include,
but are not limited to, hydroxyl, thiol, and nitrogen containing
groups such as substituted or unsubstituted amino groups and
imidazole groups. For example, residues of lysine, histidine,
and/or cysteine can be incorporated into the polycation and
introduce nucleophilic groups. An example of this is shown in FIG.
8. DOPA residue 1 can be oxidized to form a dopaquinone residue 2.
Dopaquinone is a reactive intermediate and can crosslink (i.e.,
react) with a DOPA residue on another polymer or the same polymer
to produce a di-DOPA group. Alternatively, the dopaquinone residue
can react with nucleophiles such as, for example, amino, hydroxyl,
or thiol groups via a Michael-type addition to form a new covalent
bond. Referring to FIG. 8, a lysyl group, cysteinyl group, and
histidyl group react with the dopaquinone residue to produce new
covalent bonds. Although DOPA is a suitable crosslinking group,
other groups such as, for example, tyrosine can be used herein. The
importance of crosslinking with respect to the use of the adhesive
complex coacervates described herein will be discussed below.
In other aspects, the crosslinkers present on the polycation and/or
polyanion can form coordination complexes with transition metal
ions. For example, a transition metal ion can be added to a mixture
of polycation and polyanion, where both polymers contain
crosslinkers capable of coordinating with the transition metal ion.
The rate of coordination and dissociation can be controlled by the
selection of the crosslinker, the transition metal ion, and the pH.
Thus, in addition to covalent crosslinking as described above,
crosslinking can occur through electrostatic, ionic, or other
non-covalent bonding. Transition metal ions such as, for example,
iron, copper, vanadium, zinc, and nickel can be used herein.
The polycation and polyanion are generally composed of a polymer
backbone with a plurality of chargeable groups at a particular pH.
The groups can be pendant to the polymer backbone and/or
incorporated within the polymer backbone. The polycation is any
biocompatible polymer possessing cationic groups or groups that can
be readily converted to cationic groups by adjusting the pH. In one
aspect, the polycation is a polyamino compound. The amino group can
be branched or part of the polymer backbone. The amino group can be
a primary, secondary, or tertiary amino group that can be
protonated to produce a cationic ammonium group at a selected pH.
For example, the amino group can be derived from a residue of
lysine, histidine, or imidazole attached to the polycation. Any
anionic counterions can be used in association with the cationic
polymers. The counterions should be physically and chemically
compatible with the essential components of the composition and do
not otherwise unduly impair product performance, stability or
aesthetics. Non-limiting examples of such counterions include
halides (e.g., chloride, fluoride, bromide, iodide), sulfate and
methylsulfate.
The polycation can be a synthetic polymer or naturally-occurring
(i.e., produced from organisms). In one aspect, when the polycation
is naturally-occurring, the polycation is a positively-charged
protein produced from P. californica. FIGS. 2-6 show the protein
sequences of several cement proteins produced by P. californica
(Zhao et al. "Cement Proteins of the tube building polychaete
Phragmatopoma californica" J. Biol. Chem. (2005) 280: 42938-42944).
Table 1 provides the amino acid mole % of each protein. Referring
to FIGS. 2-5, Pc1, Pc2, and Pc4-Pc8 are polycations, where the
polymers are cationic at neutral pH. The type and number of amino
acids present in the protein can vary in order to achieve the
desired solution properties. For example, referring to Table 1, Pc1
is enriched with lysine (13.5 mole %) while Pc4 and Pc5 are
enriched with histidine (12.6 and 11.3 mole %, respectively).
In the case when the polycation is a synthetic polymer, a variety
of different polymers can be used; however, it is desirable that
the polymer be biocompatible and non-toxic to cells and tissue. In
one aspect, the polycation includes a polyacrylate having one or
more pendant amino groups. For example, the backbone can be a
homopolymer or copolymer derived from the polymerization of
acrylate monomers including, but not limited to, acrylates,
methacrylates, acrylamides, and the like. In one aspect, the
backbone of the polycation is polyacrylamide. In other aspects, the
polycation is a block co-polymer, where segments or portions of the
co-polymer possess cationic groups depending upon the selection of
the monomers used to produce the co-polymer.
In one aspect, the polycation is a polyamino compound. In another
aspect, the polyamino compound has 10 to 90 mole % tertiary amino
groups. In a further aspect, the polycation polymer has at least
one fragment of the formula I
##STR00001## wherein R.sup.1, R.sup.2, and R.sup.3 are,
independently, hydrogen or an alkyl group, X is oxygen or NR.sup.5,
where R.sup.5 is hydrogen or an alkyl group, and m is from 1 to 10,
or the pharmaceutically-acceptable salt thereof. In another aspect,
R.sup.1, R.sup.2, and R.sup.3 are methyl and m is 2. Referring to
formula I, the polymer backbone is composed of
--CH.sub.2--C(R.sup.1)--C(O)X--, which is a residue of an acrylate,
methacrylate, acrylamide, or methacrylamide. The remaining portion
of formula I (CH.sub.2).sub.m--NR.sup.2R.sup.3 is the pendant amino
group. FIG. 3 (structures C and D) and FIG. 6 (4 and 7) show
examples of polycations having the fragment of formula I, where the
polymer backbone is composed acrylamide and methacrylate residues.
In one aspect, the polycation is the free radical polymerization
product of a cationic tertiary amine monomer (2-dimethylamino-ethyl
methacrylate) and acrylamide, where the molecular weight is from 10
to 20 kd and possesses tertiary monomer concentrations from 15 to
30 mol %. FIG. 4 (structures E and F) and FIG. 6 (5) provide
examples of polycations useful herein, where imidazole groups are
directly attached to the polymer backbone (structure F) or
indirectly attached to the polymer backbone via a linker (structure
E via a methylene linker).
Similar to the polycation, the polyanion can be a synthetic polymer
or naturally-occurring. In one aspect, when the polyanion is
naturally-occurring, the polyanion is a negatively-charged protein
produced from P. californica. FIGS. 2 and 7 show the sequences of
two proteins (Pc3a and Pc3b) produced by P. californica (Zhao et
al. "Cement Proteins of the tube building polychaete Phragmatopoma
californica" J. Biol. Chem. (2005) 280: 42938-42944). Referring to
Table 1, Pc3a and Pc3b are essentially composed of
polyphosphoserine, which is anionic at neutral pH.
When the polyanion is a synthetic polymer, it is generally any
biocompatible polymer possessing anionic groups or groups that can
be readily converted to anionic groups by adjusting the pH.
Examples of groups that can be converted to anionic groups include,
but are not limited to, carboxylate, sulfonate, phosphonate,
boronate, sulfate, borate, or phosphate. Any cationic counterions
can be used in association with the anionic polymers if the
considerations discussed above are met.
In one aspect, the polyanion is a polyphosphate. In another aspect,
the polyanion is a polyphosphate compound having from 10 to 90 mole
% phosphate groups. In a further aspect, the polyanion includes a
polyacrylate having one or more pendant phosphate groups. For
example, the backbone can be a homopolymer or copolymer derived
from the polymerization of acrylate monomers including, but not
limited to, acrylates, methacrylates, acrylamides, and the like. In
one aspect, the backbone of the polyanion is polyacrylamide. In
other aspects, the polyanion is a block co-polymer, where segments
or portions of the co-polymer possess anionic groups depending upon
the selection of the monomers used to produce the co-polymer. In a
further aspect, the polyanion can be heparin sulfate, hyaluronic
acid, chitosan, and other biocompatible and biodegradable polymers
typically used in the art.
In one aspect, the polyanion is a polyphosphate. In another aspect,
the polyanion is a polymer having at least one fragment having the
formula II
##STR00002## wherein R.sup.4 is hydrogen or an alkyl group, and n
is from 1 to 10, or the pharmaceutically-acceptable salt thereof.
In another aspect, wherein R.sup.4 is methyl and n is 2. Similar to
formula I, the polymer backbone of formula II is composed of a
residue of an acrylate or methacrylate. The remaining portion of
formula II is the pendant phosphate group. FIG. 7 (structure B),
shows an example of a polyanion useful herein that has the fragment
of formula II, where the polymer backbone is composed acrylamide
and methacrylate residues. In one aspect, the polyanion is the
polymerization product ethylene glycol methacrylate phosphate and
acrylamide, where the molecular weight is from 10,000 to 50,000,
preferably 30,000, and has phosphate groups in the amount of 45 to
90 mol %.
As described above, the polycation and polyanion contain
crosslinkable groups. For example, the polyanion can include one or
more groups that can undergo oxidation, and the polycation contains
on or more nucleophiles that can react with the oxidized
crosslinker to produce new covalent bonds. Polymers 3 and 7 in FIG.
6 provide examples of DOPA residues incorporated into a polyanion
and polycation, respectively. In each of these polymers, an
acrylate containing the pendant DOPA residue is polymerized with
the appropriate monomers to produce the polyanion 3 and polycation
7 with pendant DOPA residues.
It is contemplated that the polycation can be a naturally occurring
compound (e.g., protein from P. californica) and the polyanion is a
synthetic compound. In another aspect, the polycation can be a
synthetic compound and the polyanion is a naturally occurring
compound (e.g., protein from P. californica). In a further aspect,
both the polyanion and polycation are synthetic compounds.
The adhesive complex coacervates also contain one or more
multivalent cations (i.e., cations having a charge of +2 or
greater). In one aspect, the multivalent cation can be a divalent
cation composed of one or more alkaline earth metals. For example,
the divalent cation can be a mixture of Ca.sup.+2 and Mg.sup.+2. In
other aspects, transition metal ions with a charge of +2 or greater
can be used as the multivalent cation. In addition to the pH, the
concentration of the multivalent cations can determine the rate and
extent of coacervate formation. Not wishing to be bound by theory,
weak cohesive forces between particles in the fluid may be mediated
by multivalent cations bridging excess negative surface charges.
The amount of multivalent cation used herein can vary. In one
aspect, the amount is based upon the number of anionic groups and
cationic groups present in the polyanion and polycation. In the
Examples, the selection of the amount of multivalent cations with
respect to producing adhesive complex coacervates and other
physical states is addressed.
The adhesive complex coacervate can be synthesized a number of
different ways. In one aspect, the polycation, the polyanion, and
at least one multivalent cation, can be mixed with one another to
produce the adhesive complex coacervate. By adding the appropriate
amount of multivalent cation to the mixture of polyanion and
polycation, the adhesive complex coacervate can be produced. In
another aspect, the adhesive complex coacervate can be produced by
the process comprising:
(a) preparing a polyelectrolyte complex comprising admixing at
least one polycation, at least one polyanion, and at least one
multivalent cation, wherein at least one polycation or polyanion is
a synthetic compound, and the polycation and/or polyanion comprises
at least one group capable of crosslinking with each other; and (b)
adjusting the pH of the polyelectrolyte complex, the concentration
of at least one multivalent cation, or a combination thereof to
produce the adhesive complex coacervate. In this aspect, the
polyelectrolyte complex is converted to the adhesive complex
coacervate. Methods for producing the adhesive complex coacervate
in situ are described below.
The adhesive complex coacervates described herein have numerous
benefits with respect to their use as biological cements and
delivery devices. For example, the coacervates have low initial
viscosity, specific gravity greater than one, and being mostly
water by weight, low interfacial tension in an aqueous environment,
all of which contribute to their ability to adhere to a wet
surface. An additional advantage with respect to the bonding
mechanism (i.e., crosslinking) of the adhesive complex coacervates
includes low heat production during setting, which prevents damage
to living tissue. The components can be pre-polymerized in order to
avoid heat generation by in situ exothermic polymerization. This is
due for the most part by the ability of the adhesive complex
coacervates to crosslink intermolecularly under very mild
conditions as described above.
The adhesive complex coacervates described herein can be applied to
a number of different biological substrates. The substrate can be
contacted in vitro or in vivo. The rate of crosslinking within the
adhesive complex coacervate can be controlled by for example pH and
the presence of an oxidant or other agents that facilitate
crosslinking. One approach for applying the adhesive complex
coacervate to the substrate can be found in FIG. 9. The techniques
depicted in FIG. 9 are referred to herein as "spot welding," where
the adhesive complex coacervate is applied at distinct and specific
regions of the substrate. In one aspect, the adhesive complex
coacervate can be produced in situ. Referring to FIG. 9A, a
pre-formed stable PEC solution 1 composed of polycations and
polyanions at low pH (e.g., 5) is simultaneously applied to a
substrate with a curing solution 2 composed of an oxidant at a
higher pH (e.g., 10) with the use of syringes. Upon mixing, the
curing solution simultaneously produces the adhesive complex
coacervate by crosslinking the polymers on the surface of the
substrate.
In another aspect, referring to FIG. 9B, a solution of polyanions 3
and polycations 4 are applied simultaneously to the substrate. One
of the solutions has a pH higher than the other in order to produce
the adhesive complex coacervate. Referring to FIG. 9B, polyanion 3
is at a lower pH than the polycation solution 4; however, it is
also contemplated that the polyanion can be in solution having a
higher pH than the polycation. The solution having the higher pH
can include an oxidant in order to facilitate crosslinking.
FIG. 9C depicts another aspect of spot welding. In this aspect, the
substrate is primed with polycation at a particular pH. Next, a
solution of the polyanion at a higher pH is applied to the
polycation in order to produce the adhesive complex coacervate in
situ. It is also contemplated that the substrate can be primed with
polyanion first followed by polycation. An oxidant can then be
applied separately on the complex coacervate to facilitate
crosslinking to produce the adhesive complex coacervate.
Alternatively, the solution applied after the substrate has been
primed can contain the oxidant so that the adhesive complex
coacervate is formed and subsequently crosslinked in situ.
The adhesive complex coacervates described herein can be used to
repair a number of different bone fractures and breaks. The
coacervates adhere to bone (and other minerals) through several
mechanisms (see FIG. 1C). The surface of the bone's hydroxyapatite
mineral phase (Ca.sub.5(PO.sub.4).sub.3(OH)) is an array of both
positive and negative charges. The negative groups present on the
polyanion (e.g., phosphate groups) can interact directly with the
positive surface charges or it can be bridged to the negative
surface charges through the cationic groups on the polycation
and/or multivalent cations. Likewise, direct interaction of the
polycation with the negative surface charges would contribute to
adhesion. Additionally, when the polycation and/or polyanion
contain catechol moieties, they can facilitate the adhesion of the
coacervate to readily wet hydroxyapatite. Other adhesion mechanisms
include direct bonding of unoxidized crosslinker (e.g., DOPA or
other catechols) to hydroxyapatite. Alternatively, oxidized
crosslinkers can couple to nucleophilic sidechains of bone matrix
proteins.
Examples of such breaks include a complete fracture, an incomplete
fracture, a linear fracture, a transverse fracture, an oblique
fracture, a compression fracture, a spiral fracture, a comminuted
fracture, a compacted fracture, or an open fracture. In one aspect,
the fracture is an intra-articular fracture or a craniofacial bone
fracture. Fractures such as intra-articular fractures are bony
injuries that extend into and fragment the cartilage surface. The
adhesive complex coacervates may aid in the maintenance of the
reduction of such fractures, allow less invasive surgery, reduce
operating room time, reduce costs, and provide a better outcome by
reducing the risk of post-traumatic arthritis.
In other aspects, the adhesive complex coacervates described herein
can be used to join small fragments of highly comminuted fractures.
In this aspect, small pieces of fractured bone can be adhered to an
existing bone. It is especially challenging to maintain reduction
of the small fragments by drilling them with mechanical fixators.
The smaller and greater number of fragments the greater the
problem. In one aspect, the adhesive complex coacervate or
precursor thereof may be injected in small volumes to create spot
welds as described above in order to fix the fracture rather than
filling the entire crack. The small biocompatible spot welds would
minimize interference with healing of the surrounding tissue and
would not necessarily have to be biodegradable. In this respect it
would be similar to permanently implanted hardware.
In other aspects, the adhesive complex coacervates can be used to
secure scaffolds to bone and other tissues such as, for example,
cartilage, ligaments, tendons, soft tissues, organs, and synthetic
derivatives of these materials. Using the complexes and spot
welding techniques described herein, the development of scaffolds
is contemplated. Small adhesive tacks composed of the adhesive
complex coacervates described herein would not interfere with
migration of cells or transport of small molecules into or out of
the scaffold. In certain aspects, the scaffold can contain one or
more drugs that facilitate growth or repair of the bone and tissue.
For example, the scaffold can be coated with the drug or, in the
alternative, the drug can be incorporated within the scaffold so
that the drug elutes from the scaffold over time.
The adhesive complex coacervates and methods described herein have
numerous dental applications. For example, the adhesive complex
coacervates can be used to repair breaks or cracks in teeth, for
securing crowns, or seating implants and dentures. Using the spot
weld techniques described herein, the adhesive complex coacervate
or precursor thereof can be applied to a specific points in the
mouth (e.g., jaw, sections of a tooth) followed by attaching the
implant to the substrate.
In other aspects, the adhesive complex coacervates can adhere a
metal substrate to bone. For example, implants made from titanium
oxide, stainless steel, or other metals are commonly used to repair
fractured bones. The adhesive complex coacervate or a precursor
thereof can be applied to the metal substrate, the bone, or both
prior to adhering the substrate to the bone. In certain aspects,
the crosslinking group present on the polycation or polyanion can
form a strong bond with titanium oxide. For example, it has been
shown that DOPA can strongly bind to wet titanium oxide surfaces
(Lee et al., PNAS 103:12999 (2006)). Thus, in addition to bonding
bone fragments, the adhesive complex coacervates described herein
can facilitate the bonding of metal substrates to bone, which can
facilitate bone repair and recovery.
It is also contemplated that the adhesive complex coacervates
described herein can encapsulate one or more bioactive agents. The
bioactive agents can be any drug that will facilitate bone growth
and repair when the complex is applied to the bone. The rate of
release can be controlled by the selection of the materials used to
prepare the complex as well as the charge of the bioactive agent if
the agent is a salt.
EXAMPLES
The following examples are put forth so as to provide those of
ordinary skill in the art with a complete disclosure and
description of how the compounds, compositions, and methods
described and claimed herein are made and evaluated, and are
intended to be purely exemplary and are not intended to limit the
scope of what the inventors regard as their invention. Efforts have
been made to ensure accuracy with respect to numbers (e.g.,
amounts, temperature, etc.) but some errors and deviations should
be accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. or is at ambient temperature,
and pressure is at or near atmospheric. There are numerous
variations and combinations of reaction conditions, e.g., component
concentrations, desired solvents, solvent mixtures, temperatures,
pressures and other reaction ranges and conditions that can be used
to optimize the product purity and yield obtained from the
described process. Only reasonable and routine experimentation will
be required to optimize such process conditions.
Mimetic Copolymer Synthesis and Characterization.
Pc3 Analogs.
The dopa analog monomer (dopamine methacrylamide, DMA) was prepared
by slight modification of a published procedure. (Lee B P, Huang K,
Nunalee F N, Shull K R, Messersmith P B. Synthesis of
3,4-dihydroxyphenylalanine (DOPA) containing monomers and their
co-polymerization with PEG-diacrylate to form hydrogels. J Biomater
Sci Polym Ed 2004; 15(4):449-464). Briefly, a borate-dopamine
complex was reacted at pH>9 with methacryloyl chloride. After
disrupting the borate-catechol bond by acidification, the product
was washed with ethyl acetate, recrystallized from hexane, and
verified by .sup.1H NMR (400 MHz, DMSO-TMS): d8.8-8.58 (2H,
(OH).sub.2--Ar--), 7.92 (1H, --C(.dbd.O)--NH--), 6.64-6.57 (2H,
C.sub.6H.sub.2(OH).sub.2--), 6.42 (1H,
C.sub.6H.sub.2H(OH).sub.2--), 5.61 (1H,
--C(.dbd.O)--C(--CH.sub.3).dbd.CHH), 5.30 (1H,
--C(.dbd.O)--C(--CH.sub.3).dbd.CHH), 3.21 (2H,
C.sub.6H.sub.3(OH).sub.2--CH.sub.2--CH.sub.2(NH)--C(.dbd.O)--),
2.55 (2H,
C.sub.6H.sub.3(OH).sub.2--CH.sub.2--CH.sub.2(NH)--C(.dbd.O)--),
1.84 (3H, --C(.dbd.O)--C(--CH.sub.3).dbd.CH.sub.2).
Before polymerization monoacryloxyethyl phosphate (MAEP,
Polysciences) was diluted in MeOH and extracted with hexane to
remove dienes. Copolymer 1 was prepared by mixing 90 mol % MAEP, 8
mol % DMA, 2 mol % acrylamide (Aam, Polysciences), and 0.1 mol %
FITC-methacrylamide in MeOH at a final monomer concentration of 5
wt %. Free radical polymerization was initiated with
azobisisobutyronitrile (AIBN) and proceeded at 60.degree. C. for 24
hrs in sealed ampules. A similar procedure was used to make
polymers 3-7 as shown in FIGS. 2-7. Copolymer 1 (FIG. 10) was
recovered by size exclusion chromatography (SEC) in MeOH on a
Sephadex LH-20 column (Sigma-Aldrich), concentrated by rotary
evaporation, dissolved in DI water, and freeze dried.
The MW and polydispersity index (PDI) of 1 were determined by SEC
in DMF on a PLgel column (Polymer Labs) connected to a small angle
light scattering detector (Brookhaven BI-MWA) and refractive index
monitor (Brookhaven BI-DNDC). The column was calibrated with
polystyrene standards. The MW of 1 was 245 kda with a PDI of 1.9.
The dopamine sidechain concentration and reactivity was verified by
UV/VIS spectroscopy (e.sub.280=2600 M.sup.-1 cm.sup.-1). The
phosphate sidechain concentration were determined by titration with
0.005 M NaOH using an automated titrator (Brinkmann Titrando 808).
The UV/vis spectrum of 1 contained a single absorption peak at 280
nm characteristic of the catechol form of dopamine (FIG. 10B).
Addition of a 1:1 molar ratio of NaIO.sub.4 to 1 at pH 5.0 oxidized
the dopa catechol to dopaquinone with an absorption peak near 395
nm as expected. The dopaquinone peak was stable for several hrs at
pH<5.
Pc1 Analogs.
The lysine sidechains of Pc1 were mimicked with
N-(3-aminopropyl)methacrylamide hydrochloride (APMA, Polysciences).
Copolymer 2 (FIG. 10) was synthesized by dissolving 10 mol % APMA
and 90 mol % Aam in DI water, degassing with N.sub.2 and initiating
polymerization with 2 mol % ammonium persulfate (Polysciences).
Polymerization proceeded at 50.degree. C. for 24 hrs in sealed
ampules. Polymer was recovered by dialysis against water for 3
days, and then freeze dried. The primary amine sidechain mol % was
determined by .sup.1H NMR (400 MHz, DMSO-TMS) from the ratios of d
13.45 (3H, --CH3) and d 51.04 (1H, RC(.dbd.O)CHR2). The MW and PDI
of 2 were determined by SEC in PBS (20 mM PO.sub.4, 300 mM NaCl, pH
7.2) on a Superose 6 column (Pharmacia). The column was calibrated
with poly-2-hydroxypropyl methacrylate standards. The MW of 2 was
165 kd and PDI was 2.4.
Coacervate Formation and Characterization.
A 5 wt % aqueous solution of 2 was added dropwise while stirring to
a 5 wt % aqueous solution of 1 until reaching the target
amine/phosphate ratio. Total copolymer concentration was 50 mg/ml.
After mixing for 30 min the pH was adjusted with NaOH (6M).
Compositions at pH (<4) conducive to polyelectrolyte complex
(PEC) formation were diluted to 1 mg/ml in DI H.sub.2O and the zeta
potentials and size distribution of PECs were measured on a
Zeta-Sizer 3000HS (Malvern Instruments). At higher pH, coacervated
compositions were centrifuged at 2500 rpm in a microfuge
(Eppendorf), at 25.degree. C. for 2 min to collect the coacervate
phase. The volume of both phases was measured. The coacervate
phases were freeze dried and weighed to determine their mass and
concentration.
The phase behavior of 1 and 2 mixed at a 1:1 molar ratio of
phosphate to amine sidechains (50 mg/ml combined concentration)
over the pH range 3-10 is shown in FIG. 11A. The calculated net
copolymer charge normalized to the total ionizable sidechain
concentration is shown in FIG. 11B. Ascorbate, a reductant, was
added at a 1:5 molar ratio to dopa to retard oxidation of dopa by
O.sub.2 and subsequent crosslinking at elevated pH. At low pH, the
polyelectrolytes formed a stable milky solution of colloidal
polyelectrolyte complexes (PECs). The mean diameter of the PECs at
pH 2.1, determined by dynamic light scattering, was 360 nm with a
narrow dispersity and increased to 1080 nm at pH 4.0 (FIG. 11C).
The crossover of the zeta potential from positive to negative at pH
3.6 fit well with the calculated pH dependent net charge of the
complexes (FIG. 11B). The particle size could not be measured
accurately above pH 4 because the complexes flocculated. As the net
charge increased due to the deprotonation of the phosphate
sidechains, the copolymers condensed into a dense second phase. At
pH 5.1 the separated phase had the character of a loose low density
precipitate. At pH 7.2 and 8.3 the dense phase had the character of
a cohesive liquid complex coacervate (FIG. 12). The copolymers were
concentrated about three-fold to 148 and 153 mg/ml, respectively,
in the coacervated phases. At pH 9.5 the polyelectrolyte mixture
formed a dense non-liquid ionic gel. At pH 10 the copolymers went
into solution and spontaneously crosslinked through the dopaquinone
and amine sidechains into a clear hydrogel.
Extraction of divalent cations with the chelator EDTA resulted in a
50% decrease in compressive strength of P. californica tubes, a
ten-fold decrease in adhesiveness, and collapse of the glues porous
structure. The effect of divalent cations on the phase behavior of
the mimetic polyelectrolytes was investigated by mixing 1 and 2 at
amine to phosphate sidechain ratios ranging from 1:1 to 0:1 with
divalent cation to phosphate sidechain ratios ranging from 0:1 to
1:1 to create a coacervate phase diagram (FIG. 13). The pH was
fixed at 8.2, the pH of seawater, and divalent cations were added
as a 4:1 mixture of Mg.sup.2+ and Ca.sup.2+, the approximate
Mg.sup.2+/Ca.sup.2+ ratio in the natural glue determined by
elemental analysis. The highest mass of coacervate (dark gray
squares) occurred in mixtures with higher amine to phosphate
sidechain ratios and lower divalent cation to phosphate sidechain
ratios. Mixtures with lower polyamine ratios were clear (clear
squares) even at higher divalent cation/phosphate sidechain ratios.
At higher amine/phosphate and divalent cation/phosphate ratios the
solutions were turbid (light gray squares) with slight precipitates
but much less turbid than solutions containing PECs (medium gray
squares).
Mechanical Bond Testing.
Bone test specimens, .about.1 cm.sup.3, were cut with a band saw
from bovine femur cortical bone, obtained from a local grocery
store, sanded with 320 grit sandpaper, and stored at -20.degree. C.
NaIO.sub.4 at a 1:2 molar ratio to dopa sidechains was evenly
applied to one face each of two wet bone specimens. Forty ml, a
volume sufficient to completely fill the space between 1 cm.sup.2
bone interfaces, of the test coacervate solution was applied with a
pipette, the bone specimens were pressed together squeezing out a
small excess of adhesive, clamped, and immediately wrapped in PBS
(20 mM PO.sub.4, 150 mM NaCl, pH 7.4) soaked gauze. The applied
coacervate contained ascorbate at a 1:5 molar ratio to dopa to
prevent premature crosslinking. The bonded specimens were incubated
at 37.degree. C. for at least 24 hr in a sealed container
containing soaked sponges to maintain 100% humidity. Reference
specimens were bonded with 40 ml Loctite 401 superglue in exactly
the same manner. A commercial non-medical grade cyanoacrylate was
used because there are no hard tissue medical adhesives available
for comparison. Mechanical tests were performed on a custom built
material testing system using a 1 kg load cell. The instrument was
controlled and data acquired using LabView (National Instruments).
One bone of a bonded pair was clamped laterally 1 mm from the bond
interface. The second bone was pressed with a cross-head speed of
0.02 mm/s against a dull blade positioned 1 mm lateral to the bond
interface. Bond strength tests were performed at room temperature
immediately after unwrapping the wet specimens to prevent drying.
After testing, the bonds were examined for failure mode. The bonded
area was measured by tracing an outline of the bone contact surface
on paper, cutting out the trace, and determining its area from the
weight of the paper cut-out. At least 6 specimens were tested for
each condition.
The shear modulus and strength at failure were measured with bovine
cortical bone specimens bonded while wet with the three
coacervating compositions marked with an asterisk in FIG. 13. The
coacervate density in the three compositions increased with
increasing divalent cation ratios (to 120, 125, and 130 mg/ml,
respectively). Both the modulus and bond strength of the fully
hydrated specimens increased with increasing divalent cation
concentration, reaching 37% of the strength of wet bones bonded
with a commercial cyanoacrylate adhesive (FIG. 14A). The
cyanoacrylate adhesive was used as a reference point because there
are no bone adhesives in clinical use for comparison. The strength
of the mimetic adhesive is also about 1/3 the strength of natural
P. californica glue estimated to be 350 kPa and mussel byssal glue
estimated to range from 320 to 750 kPa dependent on the season. In
almost all cases the bonds failed cohesively leaving adhesive on
both bone interfaces, which suggested the compositions formed
strong interfacial bonds with hydroxyapatite. The bonds were
dimensionally stable, neither shrinking nor swelling appreciably
after complete submersion in PBS pH 7.2 for several months (FIG.
14B). Dimensional stability during cure and long term exposure to
water is an important requirement for a useful bone adhesive.
Dopamine-Mediated Copolymer Crosslinking.
Addition of NaIO.sub.4 to solutions of 3 at a 1:1 molar ratio
immediately and quantitatively oxidized DOPA (280 nm) to
dopaquinone (392 nm). Within a few minutes the quinine peak decayed
into broad general absorption as the reactive quinones formed
covalent diDOPA crosslinks (FIG. 15, top left). Crosslinking
between the quinones and primary amines (FIG. 15, bottom left) led
to a broader general absorption than diDOPA crosslinking Dopamine
oxidation and crosslinking chemistry therefore behaved as expected
in the dopamine copolymers. The dopamine copolymers rapidly formed
hydrogels as a result of oxidative crosslinking (FIGS. 15,
A&C). Oxidized phosphodopamine 3 did not gel by itself (FIG.
15B) but when mixed with 4 it gelled rapidly (FIG. 15D).
Intermolecular diDOPA crosslinking between PO.sub.4 copolymers was
inhibited but not intermolecular DOPA-amine crosslinking. This
provides a crosslinking control mechanism that may be useful for
formulating and delivering a synthetic adhesive.
pH Triggered DOPA-Mediated Crosslinking.
To explore, the pH dependence and kinetics of DOPA oxidation,
crosslinking of the dopamine copolymers were evaluated by UV-Vis
spectroscopy. Results with p(EGMP[92]-DMA[8]) (3) are shown in FIG.
16. UV-vis spectra were acquired at increasing time after addition
of a stoichiometric amount of NaIO.sub.4. At pH 5.0 (top),
dopaquinone absorbance (398 nm) was maximal in .about.15 min and
remained stable for several hrs (inset). At pH 6.0, absorbance at
398 nm peaked in <1 min and evolved into broad absorbance with
peaks at 310 and 525 nm. The broad absorbance is not due to
dopaquinone crosslinking since gels do not form (FIG. 16). For
comparison, 6 was oxidized at low pH crosslinked but at a
significantly slower rate (not shown).
The results show that the dopaquinone is stable at low pH and
diDOPA crosslinking was inhibited at higher pH in the
phosphodopamine copolymers. In the presence of the polyamine, the
covalent crosslinking was channeled toward intermolecular
amine-DOPA bonds. This is an important observation because it lays
out a path to controlled delivery and setting of the synthetic
adhesive.
In Vitro Cytotoxicity.
Solutions of 3 and 4, 40 wt % each, were mixed at low pH to form a
polyelectrolyte complex. The solution was partially oxidized with
NaIO.sub.4 and basified with NaOH just before application to
sterile glass coverslips. The adhesive-treated coverslips were
placed in the bottom of culture plate wells and human foreskin
fibroblasts, human tracheal fibroblasts, and rat primary astrocytes
in serum containing media were added to separate wells at 30K
cells/well (FIG. 17). After 24 hr, the cells were fixed with 4%
para-formaldehdye, then immunostained for the intermediate filament
protein, vimentin, to visualize cell morphology (green, A-B),
pericellular fibronectin to assess ECM secretion (red, B), glial
fibrillary protein to visual primary astrocyte morphology (green,
C), and DAPI to visualize nuclei (blue, C). The granular globs of
adhesive auto-fluoresced orangish-red (A-C).
In the representative images (FIG. 17), all cell types had
morphologies indistinguishable from cells growing on glass without
adhesive. The cells had normal motility and in several cases
extended processes that directly contacted the adhesive. No
toxicity was apparent.
Rat Calvarial Defect Model.
Production of the fragmented defect and repair with an adhesive
complex coacervate is shown in FIGS. 18A-F. Male Sprague Dawley
rats (256-290 g) (Harlan) were anesthetized with a mixture of
ketamine (65 mg/kg), xylazine (7.5 mg/kg), and acepromazine (0.5
mg/kg). At full depth of anesthesia, the eyes were covered with
ophthalmic ointment, the head shaved, and the scalp disinfected
with isopropanol and butadiene. With the prepped rats in a
stereotactic frame, a compressed air-driven drill operating at
.about.5000 RPM was lowered using a stereotactic fine toothed
manipulator. Sterile saline or PBS was continuously applied at the
craniotomy site while the custom made trephine tool was lowered 600
microns (previously determined as the skull thickness of rats the
age of which were used in the experiment). The result is a round,
accurate hole through the skull with little observable effect on
the underlying dura or vasculature (FIG. 18A-B). The bone plug was
recovered with fine curved forceps and broken into fragments using
a hemostat and fine rongeur (FIG. 18B). The bone fragments were
returned to the defect (FIG. 18C) and 5 .mu.l of test adhesive (3
and 4 mixed immediately prior to the application of the fracture)
was applied with a micropipettor (FIG. 18D). The low viscosity
adhesive solution (pre-formed PECS mixed with curing solution just
before delivery) readily and cleanly wicked into the fractures.
Within 5 min the fragments were sufficiently fixed that they could
be tapped sharply with the forceps without displacement. The
adhesive continued to turn dark reddish brown as it cured (FIG.
18E-F).
Throughout this application, various publications are referenced.
The disclosures of these publications in their entireties are
hereby incorporated by reference into this application in order to
more fully describe the compounds, compositions and methods
described herein.
Various modifications and variations can be made to the compounds,
compositions and methods described herein. Other aspects of the
compounds, compositions and methods described herein will be
apparent from consideration of the specification and practice of
the compounds, compositions and methods disclosed herein. It is
intended that the specification and examples be considered as
exemplary.
SEQUENCE LISTINGS
1
191191PRTPhragmatopoma californica 1Met Lys Val Phe Ile Val Leu Ala
Leu Val Ser Ala Ala Tyr Gly Cys1 5 10 15Gly Val Gly Ile Gly Cys Ala
Gly Gly Arg Cys Gly Gly Ala Cys Gly 20 25 30Gly Lys Gly Tyr Gly Tyr
Gly Gly Lys Leu Gly Tyr Gly Ala Tyr Gly 35 40 45Lys Gly Gly Ile Gly
Gly Tyr Gly Tyr Gly Lys Gly Cys Val Gly Gly 50 55 60Tyr Gly Tyr Gly
Gly Leu Gly Ala Gly Lys Leu Gly Gly Tyr Gly Tyr65 70 75 80Gly Gly
Ser Lys Cys Gly Gly Tyr Gly Tyr Gly Gly Gln Lys Leu Gly 85 90 95Gly
Tyr Gly Tyr Gly Gly Lys Lys Leu Gly Gly Tyr Gly Tyr Ala Ala 100 105
110Lys Lys Val Gly Gly Tyr Gly Tyr Gly Ala Lys Lys Val Gly Gly Tyr
115 120 125Gly Tyr Gly Ala Lys Lys Val Gly Gly Tyr Gly Tyr Gly Ala
Lys Lys 130 135 140Val Gly Gly Tyr Gly Tyr Gly Ala Lys Lys Val Gly
Gly Tyr Gly Tyr145 150 155 160Gly Ala Lys Lys Val Gly Gly Tyr Gly
Tyr Gly Ala Lys Lys Val Gly 165 170 175Gly Tyr Gly Tyr Gly Val Lys
Lys Val Gly Gly Tyr Gly Tyr Gly 180 185 1902210PRTPhragmatopoma
californica 2Met Lys Val Leu Ile Phe Leu Ala Thr Val Ala Ala Val
Tyr Gly Cys1 5 10 15Gly Gly Ala Gly Gly Trp Arg Ser Gly Ser Cys Gly
Gly Arg Trp Gly 20 25 30His Pro Ala Val His Lys Ala Leu Gly Gly Tyr
Gly Gly Tyr Gly Ala 35 40 45His Pro Ala Val His Ala Ala Val His Lys
Ala Leu Gly Gly Tyr Gly 50 55 60Ala Gly Ala Tyr Gly Ala Gly Ala Trp
Gly His Pro Ala Val His Lys65 70 75 80Ala Leu Gly Gly Tyr Gly Ala
Gly Ala Trp Gly His Pro Ala Val His 85 90 95Lys Ala Leu Gly Gly Tyr
Gly Gly Tyr Gly Ala His Pro Ala Val His 100 105 110Val Ala Val His
Lys Ala Leu Gly Gly Tyr Gly Ala Gly Ala Cys Gly 115 120 125His Lys
Thr Gly Gly Tyr Gly Gly Tyr Gly Ala His Pro Val Ala Val 130 135
140Lys Ala Ala Tyr Asn His Gly Phe Asn Tyr Gly Ala Asn Asn Ala
Ile145 150 155 160Lys Ser Thr Lys Arg Phe Gly Gly Tyr Gly Ala His
Pro Val Val Lys 165 170 175Lys Ala Phe Ser Arg Gly Leu Ser His Gly
Ala Tyr Ala Gly Ser Lys 180 185 190Ala Ala Thr Gly Tyr Gly Tyr Gly
Ser Gly Lys Ala Ala Gly Gly Tyr 195 200 205Gly Tyr
2103151PRTPhragmatopoma californica 3Met Lys Leu Leu Ser Val Phe
Ala Ile Val Val Leu Ala Val Tyr Ile1 5 10 15Thr His Val Glu Ala Asp
Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser 20 25 30Ser Tyr Ser Ser Ser
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr 35 40 45Ser Ser Ser Ser
Ser Tyr Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser 50 55 60Ser Ser Ser
Tyr Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Tyr Ser65 70 75 80Ser
Ser Ser Tyr Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ile Leu Thr 85 90
95Ser Thr Ser Ser Ser Asp Trp Lys Arg Lys Val Pro Ala Arg Arg Val
100 105 110Leu Arg Thr Arg Arg Phe Leu Lys Cys Val Thr Arg Cys Thr
Leu Arg 115 120 125Cys Ile Leu Phe Arg Ser Ala Lys Thr Cys Ala Arg
Lys Cys Ser Arg 130 135 140Arg Cys Leu Lys Arg Val Phe145
1504342PRTPhragmatopoma californica 4Met Lys Ser Phe Thr Ile Phe
Ala Ala Ile Leu Val Ala Leu Cys Tyr1 5 10 15Ile Gln Ile Ser Glu Ala
Gly Cys Cys Lys Arg Tyr Ser Ser Ser Ser 20 25 30Tyr Ser Ser Ser Ser
Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser 35 40 45Ser Ser Ser Ser
Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser 50 55 60Ser Ser Ser
Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser65 70 75 80Tyr
Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser 85 90
95Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser
100 105 110Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser
Tyr Ser 115 120 125Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser
Ser Ser Ser Ser 130 135 140Tyr Ser Ser Ser Ser Ser Ser Ser Tyr Ser
Ser Ser Ser Ser Ser Ser145 150 155 160Ser Ser Ser Tyr Ser Ser Ser
Ser Ser Ser Tyr Ser Ser Ser Ser Ser 165 170 175Ser Ser Ser Ser Tyr
Ser Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser 180 185 190Ser Ser Ser
Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Tyr Ser Ser 195 200 205Ser
Ser Ser Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser 210 215
220Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Tyr Ser Ser Ser
Ser225 230 235 240Ser Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser
Ser Ser Ser Tyr 245 250 255Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser
Tyr Ser Ser Ser Ser Ser 260 265 270Ser Ser Ser Ser Ser Ser Tyr Ser
Ser Ser Ser Ser Ser Tyr Ser Ser 275 280 285Ser Ser Ser Ser Ser Tyr
Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 290 295 300Tyr Ser Ser Ser
Ser Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser305 310 315 320Ser
Ser Ser Ser Tyr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 325 330
335Ser Ser Tyr Ser Ser Ser 3405272PRTPhragmatopoma californica 5Met
Pro Thr Leu Tyr Lys Lys Val Gly Lys Leu Val Ile Leu Ala Ile1 5 10
15Ile Val Thr Val Ala Ser Val Ala Ser Ala Gly Tyr Pro Thr Tyr Ser
20 25 30Pro Ser Gly Gly Thr His Ser Gly Tyr Asn Gly Pro His Gly Asn
Val 35 40 45Val Lys Lys Thr Tyr Arg Gly Pro Tyr Gly Ala Gly Ala Ala
Lys Ala 50 55 60Trp Asn Gly Tyr His Gly Ala Gly Tyr Thr Ser Val His
His Gly Pro65 70 75 80Ala Ser Thr Ser Trp His Thr Ser Trp Ser Asn
Lys Lys Gly Gly Tyr 85 90 95Gly Tyr Gly Leu Lys Asn Lys Gly Tyr Gly
Tyr Gly Leu Lys Lys Val 100 105 110Gly Tyr Gly Val Gly Leu His Ala
Ala Gly Trp His Gly Val Gly Pro 115 120 125Tyr Gly Ala Gly Tyr His
Gly Ala Gly Trp Asn Gly Leu Gly Tyr His 130 135 140Gly Ala Gly Tyr
Gly Val His Gly Val Gly Leu His Gly Ala Gly Tyr145 150 155 160Gly
Leu His Gly Val Gly Leu His Gly Val Gly Tyr Gly Leu His Gly 165 170
175Val Gly Leu His Gly Ala Gly Tyr Gly Leu His Gly Val Gly Leu His
180 185 190Gly Ala Gly Tyr Gly Ile His Gly Val Gly Leu His Gly Ala
Gly Tyr 195 200 205Gly Ile His Gly Val Gly Leu His Gly Val Gly Tyr
Gly Leu His Gly 210 215 220Val Gly Leu His Gly Ala Gly Tyr Gly Leu
His Gly Val Gly Leu His225 230 235 240Gly Ala Gly Tyr Gly Ile His
Gly Val Gly Leu His Gly Ala Gly Cys 245 250 255Gly Ile His Lys Thr
Ala Cys Tyr Gly Val Gly Leu His Gly His Tyr 260 265
2706144PRTPhragmatopoma californica 6Met Lys Phe Leu Val Leu Leu
Ala Leu Val Ala Ser Ala Ser Ala Tyr1 5 10 15Tyr Pro Leu Met Gly Gly
Phe His Gly Gly Trp His Ala Pro Met Val 20 25 30His Gly Gly Leu Tyr
His Gly Gly Trp His Ala Pro Met Val His Gly 35 40 45Gly Leu Tyr His
Gly Gly Trp His Ala Pro Ile Val His Gly Gly Trp 50 55 60His Ala Pro
Val Phe His Ala Pro Ala Pro Ile His Thr Val Ser His65 70 75 80Ser
Val Val Asn His Val Pro Met Met Pro Met Trp His His Pro Ala 85 90
95Pro Ala Pro Ala Pro Ala Pro Arg Pro Gly Arg Thr Ile Ile Leu Gly
100 105 110Gly Gly Lys Tyr Gly Pro Phe Gly Lys Tyr Gly Gly Gly Ala
Gly Leu 115 120 125Leu Ala Leu Gly Ala Leu Gly Gly Asn Gly Gly Phe
Trp Lys Arg Arg 130 135 1407328PRTPhragmatopoma californica 7Met
Leu Phe Tyr Asn Ala Asn Phe Val Gln Lys Ser Trp Val Leu Ile1 5 10
15Leu Leu Gly Leu Ala Ala Val Val Ala Cys Ser Glu Tyr Asp Lys Gly
20 25 30Leu Gly Gly Tyr Gly Arg Pro Ser Tyr Gly Gly Arg Arg Gly Tyr
Gly 35 40 45Gly Arg Arg Gly Leu Gln Tyr His Gly Lys Tyr Gln Gly Arg
Cys Glu 50 55 60Tyr Asp Gly Leu Tyr Phe Arg Asp Glu Lys Ser Phe Val
Tyr Cys Ser65 70 75 80Asn Arg Asn Ser Tyr Ile Gln Pro Cys Ala Pro
Gly Thr Arg Asn Ser 85 90 95Pro Tyr Thr Lys Tyr Asn Arg Gly Ser Lys
Tyr Asn Tyr Arg Asp Phe 100 105 110Cys Glu Val Asn Leu Val Asp Ser
Gly Tyr Val Pro Lys Pro Gly Tyr 115 120 125Leu Pro Ala Pro Lys Lys
Ala Tyr Pro Thr Lys Val Tyr Asp Leu Lys 130 135 140Val Asp Tyr Ala
Pro Lys Val Asp Tyr Ala Pro Lys Val Asp Tyr Ala145 150 155 160Pro
Lys Val Asp Tyr Ala Pro Lys Val Asp Tyr Val Ala Pro Lys Ala 165 170
175Ser Tyr Val Pro Pro Lys Ala Ser Tyr Val Asp Pro Thr Pro Thr Tyr
180 185 190Gly Tyr Glu Ala Pro Phe Lys Gly Gly Tyr Asp Lys Pro Ser
Tyr Gly 195 200 205Lys Asp Val Asp Thr Ser Tyr Glu Ser Lys Thr Thr
Tyr Thr Val Glu 210 215 220Lys Thr Ala Asp Lys Gly Tyr Gly Lys Gly
Tyr Gly Asp Lys Glu Ile225 230 235 240Ser Ala Lys Lys Ser Tyr Thr
Leu Thr Glu Lys Arg Asp Tyr Asp Thr 245 250 255Gly Tyr Asp Asn Ser
Arg Ser Asp Glu Asp Ser Lys Glu Tyr Gly Tyr 260 265 270Asp Asn Asp
Arg Ser Glu Ser Tyr Glu Arg Thr Glu Ser Tyr Thr Asp 275 280 285Glu
Arg Thr Asp Gly Tyr Gly Thr Gln Lys Val Glu Tyr Thr Gln Gln 290 295
300Ser Glu Tyr Asp Arg Val Thr Arg Arg Gly Ile Trp Leu His Lys
Gly305 310 315 320Thr Glu Val Glu His Val Leu Tyr
3258136PRTPhragmatopoma californicamisc_feature(49)..(49)Xaa can be
any naturally occurring amino acid 8Met Asn Thr Phe Val Val Leu Ala
Ala Ile Val Ala Val Ala Ala Cys1 5 10 15Ser Gly Gly Tyr Asp Gly Arg
Gln Tyr Thr Tyr Arg Gly Arg Tyr Asn 20 25 30Asn Lys Cys Gly Asn Asp
Gly Leu Tyr Phe Lys Asp Asp Lys Asn Phe 35 40 45Xaa Phe Cys Ser Asn
Gly Asn Ser Tyr Val Gln Pro Cys Ala Pro Gly 50 55 60Thr Arg Asn Ser
Gly Tyr Asn Asn Tyr Lys Gln Gly Ser Ile Tyr Asn65 70 75 80Tyr Arg
Asp Phe Cys Asp Val Asn Leu Val Asp Glu Gly Tyr Gly Val 85 90 95Gly
Ala Lys Pro Gly Tyr Asn Lys Gly Tyr Asn Pro Gly Tyr Asn Pro 100 105
110Gly Tyr Gly Gly Tyr Asn Pro Gly Tyr Ser Thr Gly Tyr Gly Gly Tyr
115 120 125Lys Ala Gly Pro Gly Pro Tyr Trp 130
1359158PRTPhragmatopoma californica 9Met Lys Leu Ala Leu Leu Leu
Leu Val Ala Val Cys Ala Ala Val Ala1 5 10 15Val Asn Ala Cys Gly Pro
Leu Gly Cys Ser Gly Gly Tyr Gly Gly Val 20 25 30Leu Lys Cys Gly Val
Gly Gly Cys Ala Leu Gly Gly Tyr Gly Gly Gly 35 40 45Tyr Ser Ala Gly
Ile Gly Gly Tyr Gly Ile Lys Arg Leu Gly Cys Arg 50 55 60Gly Gly Arg
Cys Gly Leu Arg Arg Arg Val Gly Cys Arg Gly Gly Arg65 70 75 80Cys
Gly Leu Arg Gly Arg Leu Gly Cys Arg Gly Gly Arg Cys Gly Leu 85 90
95Arg Lys Leu Gly Cys Arg Gly Gly Arg Cys Gly Leu Arg Gly Arg Leu
100 105 110Gly Cys Arg Gly Gly Arg Cys Gly Leu Arg Lys Arg Leu Gly
Cys Arg 115 120 125Gly Gly Arg Cys Gly Arg Gly Gly Tyr Gly Gly Gly
Tyr Gly Gly Val 130 135 140Cys Ser Lys Gly Val Cys Gly Gly Tyr Pro
Ala Tyr Gly Lys145 150 15510236PRTPhragmatopoma californica 10Met
Lys Val Ser Ile Ala Val Leu Ile Met Cys Cys Ile Ala Ala Val1 5 10
15Leu Ala Asp Gly Tyr Lys Ser Lys Asn Gly Gly Gln Ala Gly Gly Tyr
20 25 30Gly Gly Tyr Gly Ser Gly Tyr Gly Gly Gly Tyr Gly Gly Gly Tyr
Asp 35 40 45Gly Gly Tyr Gly Gly Glu Lys Gly Lys Ser Gly Lys Gly Tyr
Gly Asp 50 55 60Arg Lys Gly Lys Ser Glu Lys Gly Tyr Gly Asn Gly Lys
Gly Lys Gly65 70 75 80Gly Ser Gly Tyr Gly Gly Gly Tyr Asp Gly Gly
Tyr Gly Gly Gly Lys 85 90 95Gly Lys Ser Gly Ser Gly Tyr Gly Gly Gly
Tyr Asp Gly Gly Tyr Gly 100 105 110Gly Gly Lys Gly Lys Ser Gly Ser
Gly Tyr Gly Gly Gly Tyr Asp Gly 115 120 125Gly Tyr Asp Gly Gly Tyr
Gly Gly Gly Lys Gly Lys Ser Gly Ser Gly 130 135 140Phe Gly Gly Gly
Tyr Asp Gly Gly Tyr Asp Gly Gly Tyr Gly Gly Gly145 150 155 160Lys
Gly Lys Ser Gly Ser Gly Tyr Gly Gly Gly Tyr Asp Gly Gly Tyr 165 170
175Asp Gly Gly Tyr Gly Gly Gly Lys Gly Lys Ser Gly Ser Gly Tyr Gly
180 185 190Gly Gly Tyr Asp Gly Gly Tyr Asp Gly Gly Tyr Gly Gly Gly
Lys Gly 195 200 205Lys Ser Gly Ser Gly Tyr Gly Gly Gly Tyr Asp Gly
Gly Tyr Asp Gly 210 215 220Arg Tyr Gly Gly Gly Lys Gly Lys Ser Gly
Ser Gly225 230 23511191PRTPhragmatopoma californica 11Met Lys Leu
Ile Cys Leu Val Leu Leu Ala Val Cys Ile Val Ala Val1 5 10 15Ser Ala
Ser Ser Ser Ser Gly Gly Arg Arg Arg Arg Val Ile Val Ile 20 25 30Gly
Asn Arg Gly Arg Ala Pro Ala Arg Pro Arg Ser Asn Ile His Tyr 35 40
45His Met His Ala Pro Gln Pro Arg Met Met Met Ala Pro Arg Met Met
50 55 60Met Ala Pro Met Met Met Ala Pro Met Ala Met Pro Ala Thr Ser
His65 70 75 80Val Tyr Gln Ser Val Ser Tyr Pro Gly Ala Met Tyr Arg
Tyr Gly Leu 85 90 95Gly Ser Leu Gly Gly Gly Phe Ile Ser Gly Gly Leu
Gly Gly Ile Val 100 105 110Gly Gly Gly Leu His Gly Gly Val Val Thr
Ser Gly Leu His Gly Gly 115 120 125Val Val Thr Ser Gly Leu His Gly
Gly Val Val Thr Ser Gly Leu His 130 135 140Gly Gly Leu Val Ser Gly
Gly Trp His Ser Gly Val Val Ser His Gly145 150 155 160Gly Leu Ile
Gly Gly Gly Ile His Thr Thr Tyr Gly Gly Phe His Lys 165 170 175Gly
Val Val His Gly Gly Tyr Thr Gly His Tyr Gly Lys Arg Arg 180 185
19012102PRTPhragmatopoma californica 12Met Lys Leu Ala Val Phe Ala
Leu Leu Val Ala Phe Ala Ile Val Tyr1 5 10 15Thr Ala Glu Gly Leu Val
Tyr Gly Gly Gln Lys Gly Tyr Gly Tyr Gly 20 25 30Gly Lys Gly Tyr Gly
Tyr Gly Cys Thr Gly Gly Tyr Gly Leu Tyr Gly 35 40 45Gly Lys Gly Tyr
Gly Tyr Gly Lys Gly Tyr Gly Tyr Gly Cys Arg Gly 50 55
60Gly Tyr Gly Tyr Gly Lys Gly Tyr Gly Tyr Gly Gly Lys Tyr Arg Gly65
70 75 80Tyr Gly Tyr Gly Asn Lys Val Gly Tyr Gly Tyr Gly Gln Gln Leu
Gly 85 90 95Tyr Lys Asn Gly Arg Lys 1001391PRTPhragmatopoma
californica 13Leu Asp Gly Gly Cys Lys Pro Thr Gly Gly Phe Ile Lys
Gly Ser Val1 5 10 15Gly Pro Cys Gly Gly Tyr Asn His Gln His Val Val
Gly Pro Asn Gly 20 25 30Ala His Gly Arg Arg Val Gly Tyr Gly Pro Asn
Gly Lys Tyr Ser Gln 35 40 45Ile Tyr Gly Asn Gly Pro Gly Gly Arg Tyr
Ser His Thr Val Val Tyr 50 55 60Pro Arg Val Arg Pro Tyr Gly Gly Tyr
Gly Phe Lys Gly Gly Tyr Gly65 70 75 80Gly Tyr His Gly Val Gly Tyr
Lys Gly Gly Tyr 85 9014145PRTPhragmatopoma californica 14Met Lys
Val Phe Val Ala Ala Leu Leu Leu Cys Cys Ile Ala Ala Ala1 5 10 15Ala
Ala Glu Asp Gly Tyr Gly Phe Gly Tyr Asp Gly Tyr Gly Ser Gly 20 25
30Tyr Gly Tyr Asp Gly Tyr Ser Tyr Gly Gly Asp Lys Gly Tyr Gly Tyr
35 40 45Gly Lys Gly Lys Gly Tyr Gly Tyr Glu Gly Gly Lys Gly Tyr Gly
Tyr 50 55 60Glu Gly Gly Lys Gly Tyr Gly His Glu Glu Gly Lys Gly Tyr
Gly His65 70 75 80Glu Gly Gly Lys Gly Tyr Gly Tyr Glu Gly Gly Lys
Gly Tyr Gly Tyr 85 90 95Gly Gly Gly Lys Gly Tyr Gly His Asp Gly Gly
Lys Gly Tyr Gly His 100 105 110Asp Gly Gly Lys Gly Tyr Gly Tyr Gly
Gly Gly Lys Gly Tyr Gly His 115 120 125Glu Gly Gly Lys Gly Tyr Gly
Tyr Glu Gly Gly Lys Gly Tyr Gly Lys 130 135
140Tyr14515134PRTPhragmatopoma californica 15Met Arg Ile Val Ile
Cys Leu Leu Val Leu Val Ala Gly Ala Tyr Gly1 5 10 15Ile Gly Cys Gly
Tyr Gly Gly Tyr Gly Gly Tyr Gly Gly Gly Phe His 20 25 30Gly Gly Tyr
Ile Gly Tyr His Gly Gly Tyr Pro Gly Tyr Ser Gly Gly 35 40 45Phe Arg
Gly Tyr Gly Tyr Pro Gly Arg Val His Thr Asn Val Val His 50 55 60His
Asn Ile Pro Val Phe Met Pro Pro Pro Met Pro Arg Arg Ala Pro65 70 75
80Ala Pro Ala Pro Arg Gly Arg Thr Ile Ile Leu Gly Gly Gly Lys Tyr
85 90 95Gly Leu Phe Gly Lys Lys Ser Lys Asn Lys Gly Phe Gly Gly Leu
Gly 100 105 110Val Leu Ser Leu Leu Gly Gly Leu Gly Gly Lys Gly Gly
Gly Gly Ile 115 120 125Arg Phe Leu Gly Arg Lys
13016226PRTPhragmatopoma californica 16Met Lys Val Ile Ile Leu Leu
Ala Thr Val Ala Ala Val Tyr Gly Cys1 5 10 15Gly Gly Trp Asn Gly Gly
Phe Gly Gly Gly Lys Ala Cys Gly Gly Gly 20 25 30Trp Gly Ala Lys Ala
Leu Gly Gly Tyr Gly Ser Tyr Asn Gly Asn Gly 35 40 45Tyr Gly Ala His
Pro Val Ala Val Lys Ser Ala Phe Asn Lys Gly Val 50 55 60Ser Tyr Gly
Ala Arg Ser Ala Val Lys Ala Thr Arg Gly Phe Ala Tyr65 70 75 80Gly
Lys Gly Ser Ser Tyr Gly Tyr Gly Ala His Pro Ala Val Lys Ser 85 90
95Ala Phe Gly Asn Gly Phe Lys Thr Gly Ala His Ala Ala Val Asn Gly
100 105 110Tyr Gly Tyr Gly Ala Val Lys Ser Ala Leu Ser Gly Gly Tyr
Gly Tyr 115 120 125Gly Ser Tyr Gly Ala His Pro Ala Val Lys Ser Ala
Tyr Arg Lys Gly 130 135 140Leu Ser Tyr Gly Ala Lys Ser Ala Val Lys
Ala Thr Arg Gly Phe Ala145 150 155 160Tyr Gly Arg Ser Gly Tyr Gly
Ala His Pro Val Val Lys Ser Ala Phe 165 170 175Ser Asn Gly Phe Lys
Tyr Gly Ala His Ala Ala Val Lys Ala Thr Asn 180 185 190Gly Tyr Gly
Tyr Gly Ala Val His Pro Ala Val Lys Ala Ala Val Lys 195 200 205Gly
Gly Tyr Gly Tyr Gly Asn Lys Gly Gly Tyr Gly Ala Gly Tyr Ala 210 215
220Ala Tyr2251789PRTPhragmatopoma californica 17Met Lys Val Phe Val
Ala Thr Leu Leu Leu Cys Cys Ile Ala Ala Ala1 5 10 15Ala Ala Ala Gly
Tyr Gly Asn Gly Tyr Ala Gly Tyr Gly Ser Gly Tyr 20 25 30Ala Gly Tyr
Gly Thr Gly Tyr Ala Gly Tyr Gly Ser Gly Tyr Gly Tyr 35 40 45Asp Gly
Tyr Gly Tyr Gly Gly Gly Lys Gly Tyr Gly Tyr Gly Gly Asp 50 55 60Lys
Gly Tyr Gly Tyr Gly Gly Lys Gly Tyr Gly Tyr Gly Gly Gln Lys65 70 75
80Gly Tyr Gly Tyr Gly Tyr Gly Lys Tyr 8518105PRTPhragmatopoma
californica 18Met Lys Leu Leu Leu Leu Phe Ala Leu Ala Ala Val Ala
Val Ala Leu1 5 10 15Pro Tyr Gly Tyr Ser Gly Lys Pro Gly Tyr Gly Tyr
Asp Ala Val Asp 20 25 30Ala Val Tyr Asn Arg Leu Ala Asp Lys Gln Gln
Ala Val Asn Arg Lys 35 40 45Ala Glu Tyr Val Gly Ala Gly Thr Gly Thr
Ala Lys Tyr Asn Gly Val 50 55 60Pro Gly Ala Asn Tyr Gly Tyr Glu Asn
Asp Arg Lys Tyr Gly Tyr Asp65 70 75 80Asn Lys Gly Tyr Gly Gly Tyr
Gly Asp Lys Gly Tyr Gly Gly Tyr Gly 85 90 95Asp Lys Gly Leu Tyr Asp
Gly Tyr Tyr 100 10519275PRTPhragmatopoma californica 19Lys Tyr Tyr
Asp Asp Glu Lys Arg Asp Ala Asp Lys Tyr Arg Lys Pro1 5 10 15Ser Tyr
Asn Pro Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys Lys Ile 20 25 30Tyr
Asn Asp Asp Glu Lys Arg Asp Ala Asp Gln Tyr Arg Ile Ser Tyr 35 40
45Asn Pro Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys Lys Lys Tyr Tyr
50 55 60Asp Asp Glu Lys Arg Asp Ala Tyr Lys Tyr Arg Asn Pro Ser Tyr
Asn65 70 75 80Pro Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys Lys Ile
Tyr Tyr Asp 85 90 95Asp Glu Lys Arg Asp Ala Asp Gln Tyr Arg Asn Pro
Ser Tyr Asn Pro 100 105 110Tyr Asn Thr Tyr Lys Asp Tyr Pro Pro Lys
Lys Lys Tyr Tyr Asp Asp 115 120 125Glu Lys Arg Asp Ala Asp Gln Tyr
Arg Asn Pro Ser Tyr Asn Pro Tyr 130 135 140Asn Thr Tyr Lys Asp Tyr
Leu Pro Lys Lys Lys Tyr Tyr Asp Asp Glu145 150 155 160Lys Arg Asp
Ala Asp Gln Tyr Arg Lys Pro Ser Tyr Asn Pro Tyr Asn 165 170 175Ser
Tyr Lys Asp Tyr Pro Pro Lys Lys Lys Tyr Tyr Asp Asp Glu Lys 180 185
190Arg Asp Ala Asp Gln Tyr Arg Asn Pro Ser Tyr Asn Pro Tyr Asn Thr
195 200 205Tyr Lys Asp Tyr Leu Pro Lys Lys Lys Tyr Tyr Asp Asp Glu
Lys Arg 210 215 220Asp Ala Asp Gln Tyr Arg Asn Pro Ser Tyr Asn Pro
Tyr Asn Thr Tyr225 230 235 240Lys Asp Tyr Pro Pro Lys Lys Lys Tyr
Tyr Asp Asp Glu Lys Arg Asp 245 250 255Ala Asp Gln Tyr Arg Asn Pro
Ser Tyr Asn Pro Tyr Asn Thr Tyr Lys 260 265 270Asp Tyr Pro 275
* * * * *
References